title: Toxicology and carcinogenesis studies of isoeugenol (CAS No. 97-54-1) in F344/N rats and B6C3F1 mice (gavage studies).
abstract: Isoeugenol is one of several structurally similar phenylpropenoid compounds produced by plants. It has been extracted from calamus, savory, basil, ylang-ylang, clove, tuberose, jonquil, nutmeg, tobacco, sandalwood, dill seed, mace, gardenia, petunia, and other flowers. Isoeugenol can also be produced by isomerization of eugenol, which occurs naturally in clove, pimento, bay leaf, and cinnamon. As a fragrance with a spicy, carnation-like odor, isoeugenol is incorporated into numerous household and personal hygiene products, including perfumes, cream lotions, soaps, and detergents. As a flavoring agent, isoeugenol is added to nonalcoholic drinks, baked foods, and chewing gums. Isoeugenol was nominated by the National Cancer Institute and was selected for carcinogenicity testing because of widespread human exposure through its use as a flavoring and fragrance agent and because of its structural similarity to phenylpropenoids such as safrole, isosafrole, eugenol, methyleugenol, estragole, and anethole, most of which are known rodent carcinogens. Male and female F344/N rats and B6C3F1 mice were administered isoeugenol (99% or greater pure) in corn oil by gavage for 3 months or 2 years. Genetic toxicity tests were conducted in Salmonella typhimurium, Escherichia coli, cultured Chinese hamster ovary cells, and mouse peripheral blood erythrocytes. 3-MONTH STUDY IN RATS: Groups of 10 male and 10 female rats were exposed to isoeugenol in corn oil by gavage at doses of 0, 37.5, 75, 150, 300, or 600 mg/kg, 5 days per week for 14 weeks. All rats survived to the end of the study except one 600 mg/kg male and one 37.5 mg/kg female that were killed in dosing accidents. Mean body weights of all exposed groups of males were significantly less than that of the vehicle control group; however, only the decrease for the 600 mg/kg group exceeded 10% and was considered related to isoeugenol exposure. Liver weights were significantly increased in 300 and 600 mg/kg females. The incidences of minimal atrophy of the olfactory epithelium of the nose were significantly increased in 150 mg/kg or greater males and in 300 or 600 mg/kg females. The incidence of atrophy of olfactory nerve bundles was significantly increased in 600 mg/kg females. Minimal to mild periportal hepatocellular cytoplasmic alteration occurred in all 300 or 600 mg/kg females. 3-MONTH STUDY IN MICE: Groups of 10 male and 10 female mice were exposed to isoeugenol in corn oil by gavage at doses of 0, 37.5, 75, 150, 300, or 600 mg/kg, 5 days per week for 14 weeks. All mice survived to the end of the study. The mean body weight of 600 mg/kg males was significantly less (12%) than that of the vehicle controls. Liver weights of 300 and 600 mg/kg males were significantly greater than those of the vehicle controls. Minimal to moderate atrophy of olfactory epithelial tissue and nerve bundles was observed in 600 mg/kg males and females. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were exposed to isoeugenol in corn oil by gavage at doses of 0, 75, 150, or 300 mg/kg, 5 days per week for 105 weeks. Survival rates of exposed male and female rats were similar to those of vehicle controls. Mean body weights of 300 mg/kg male rats were 9% greater than the vehicle controls at the end of the study. The general lack of toxicity and nonneoplastic lesions indicates that rats might have been able to tolerate higher doses. Two male rats in the 300 mg/kg group had rare benign or malignant thymomas, while two other males in this group had rare mammary gland carcinomas. Low incidences of minimal atrophy and minimal to mild respiratory metaplasia of the olfactory epithelium were increased in 150 mg/kg males and 300 mg/kg males and females. Similar incidences of minimal to mild olfactory epithelial degeneration in 300 mg/kg males were also increased. Incidences of keratoacanthoma of the skin were decreased in 150 and 300 mg/kg males. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were exposed to isoeugenol in corn oil by gavage at doses of 0, 75, 150, or 300 mg/kg, 5 days per week for 104 (females) or 105 (males) weeks. Survival of 300 mg/kg males was significantly decreased compared to the vehicle controls. Mean body weights of 300 mg/kg male and female groups were less than those of vehicle controls at the end of the study, 10% and 15% less, respectively. In all groups of exposed males, the incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined) were significantly greater than those in the vehicle control group; incidences of multiple hepatocellular adenoma were also significantly increased. Incidences of clear cell focus were significantly increased in 75 and 150 mg/kg male mice. There was a significant positive trend in the incidences of histiocytic sarcoma in females, and this neoplasm occurred in multiple tissues. Incidences of respiratory metaplasia in olfactory epithelium in all exposed groups and of atrophy and hyaline droplet accumulation in all exposed groups except 75 mg/kg females were significantly greater than those in corresponding vehicle control groups. Incidences of minimal to marked hyperplasia of Bowman's gland were increased significantly in all exposed groups. Incidences of minimal to mild necrosis of renal papilla and mild to moderate necrosis of renal tubules were increased significantly in 300 mg/kg females. Incidences of forestomach squamous hyperplasia, inflammation, and ulceration (males only) increased with exposure and were significant in the 300 mg/kg groups. The incidence of glandular stomach ulcers was low but significantly increased in the 300 mg/kg groups.
  
  
  RESULTS
  Isoeugenol was not mutagenic in two independent assays in bacteria (S. typhimurium and E. coli) conducted with and without exogenous metabolic activation (S9 liver enzymes). Neither did it induce chromosomal aberrations in cultured Chinese hamster ovary cells, with or without S9 activation. Frequencies of micronucleated erythrocytes were not increased in peripheral blood of male mice exposed to isoeugenol by gavage for 3 months; however, an increasing trend and a threefold increase in the 600 mg/kg group indicate a positive result for this test in female mice.
  
  
  CONCLUSIONS
  Under the conditions of these 2-year gavage studies, there was equivocal evidence of carcinogenic activity of isoeugenol in male F344/N rats based on increased incidences of rarely occurring thymoma and mammary gland carcinoma. There was no evidence of carcinogenic activity of isoeugenol in female F344/N rats administered 75, 150, or 300 mg/kg. There was clear evidence of carcinogenic activity of isoeugenol in male B6C3F1 mice based on increased incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma or carcinoma (combined). There was equivocal evidence of carcinogenic activity of isoeugenol in female B6C3F1 mice based on increased incidences of histiocytic sarcoma. Exposure to isoeugenol resulted in nonneoplastic lesions of the nose in male and female rats; of the nose, forestomach, and glandular stomach in male and female mice; and of the kidney in female mice.
mesh_terms: D000818:Animals; D001835:Body Weight / Q000187:drug effects; D015197:Carcinogenicity Tests; D004305:Dose-Response Relationship, Drug; D005054:Eugenol / Q000031:analogs & derivatives* / Q000633:toxicity; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009374:Neoplasms, Experimental / Q000139:chemically induced*; D051381:Rats; D011916:Rats, Inbred F344
chemical_list: D005054:Eugenol; C036643:isoeugenol